1. Field of the Invention
The present invention relates to mutant proteins of transglutaminase of actinomycetous origin. Transglutaminase (also simply referred to as TG) is widely utilized for food processing and the like since it forms a cross-linking bond between proteins and produces a gel-like substance. Mutant TG with improved thermal and pH stability allows for improved storage stability of TG, reactions at high temperature, expansion of applicable reaction pH range, and the like, making it possible to apply this enzyme to new fields.
2. Brief Description of the Related Art
Transglutaminase is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxamide group in a peptide chain of a protein. When this enzyme acts on a protein, reactions which result in the formation of ε-(γ-Glu)-Lys cross-linking and replacement of Gln with Glu by deamidation can occur. Transglutaminases of animal origin and those of microbial origin are known. The TG enzymes of animal origin are Ca2+-dependent, and are distributed in animal organs, skin, blood, and the like. For example, guinea pig liver transglutaminase (K. Ikura et al., Biochemistry, 27, 2898, 1988), human epidermal keratinocyte transglutaminase (M. A. Phillips et al., Proc. Natl. Acad. Sci. U.S.A, 87, 9333, 1990), human blood coagulation factor XIII (A. Ichinose et al., Biochemistry, 25, 6900, 1986), and the like exist. The TG enzymes of microbial origin are Ca2+-independent, and have been discovered in the genus Streptomyces, for example, Streptomyces griseocarneus NBRC 12776, Streptomyces cinnamoneus NBRC 12852, Streptomyces mobaraensis NBRC 13819, and the like. A transglutaminase found in a culture supernatant of a variant of Streptomyces mobaraensis is referred to as MTG (Microbial Transglutaminase). Furthermore, a Ca2+-independent transglutaminase has also been discovered in Streptomyces lydicus NRRL B-3446 (JP-A-10-504721). It has been found, as a result of peptide mapping and gene structural analysis, that the primary structures of the transglutaminases produced by these microorganisms do not have any homology with those of animal origin (EP 0 481 504 A1).
MTG is a monomeric protein consisting of 331 amino acids, and having a molecular weight of about 38,000 (T. Kanaji et al., Journal of Biological Chemistry. 268, 11565, 1993). Because MTG is produced from a culture of one of the aforementioned microorganisms, and the like, through a purifying operation, there have been problems with respect to obtaining sufficient yields, efficiency, and the like. Attempts have also been made to produce transglutaminase by genetic engineering techniques. A method based on secretory expression by Escherichia coli (E. coli), yeast, and the like (JP-A-5-199883), a method wherein Escherichia coli is allowed to express MTG as a protein inclusion body, after which this inclusion body is solubilized with a protein denaturant, treated to remove the denaturant, and then reconstituted to produce active MTG (JP-A-6-30771), and a method for secretory expression of MTG using Corynebacterium glutamicum (WO2002/081694) have been reported. Unlike transglutaminases of animal origin, MTG and other transglutaminases of microbial origin are Ca24-independent, and are hence utilized for production of gelled foods such as jellies, yoghurt, cheese, or gel cosmetics, and the like, as well as improvement of the quality of meat, and the like (JP-A-64-27471). MTG is also utilized for production of raw materials for heat-stable microcapsules, carriers for immobilized enzymes and the like, and is therefore a highly useful industrial enzyme. Regarding enzymatic reaction conditions, a gelled food, for example, does not set if the enzymatic reaction time is too short, and conversely, if the reaction time is too long, the gelled food becomes too hard to be a commercial product. Hence, when MTG is utilized for production of gelled foods such as jellies, yoghurt, cheese, or gel cosmetics and the like, as well as improvement of the quality of meat and the like, the desired product is prepared by adjusting substrate and enzyme concentrations, reaction temperature, and reaction time. However, as MTG-based foods, reagents and the like have become increasingly diverse, there have been some cases where the desired product cannot be prepared solely by adjusting concentrations, temperature, time and the like. Therefore, there is a need for modifying the enzymatic activity of MTG.
Wild-type MTG (wild-type MTG means an MTG that occurs naturally and has not undergone a modification in the amino acid sequence thereof) is known to be stable at a pH between about 4 and 10, and is usually stable over a relatively broad range of pH values, but the reaction of wild-type MTG under extremely acidic or alkaline conditions is difficult. The optimum temperature for reacting wild-type MTG is about 55° C., but such reactions are difficult due to the high temperatures. Even at lower temperatures, incubation for a long time can result in reduced enzymatic activity. Therefore, a mutant transglutaminase with improved pH stability, thermal stability and the like, if any, would enable new uses of transglutaminase.
MTG has been utilized mainly in the food area so far. Feasibility of application in a wide variety of uses, including textiles, chemical products (photographic films, tanning), feeds, cosmetics, and pharmaceuticals, has been suggested.
In the textile area, wool modification with transglutaminase is known. Specifically, it is known that by treating wool with transglutaminase, anti-shrinkage quality, anti-pilling quality and hydrophobicity can be conferred while maintaining the original texture (JP-A-3-213574). When transglutaminase is used for wool, a reaction to keratin at high temperature in a short time, if possible, would increase throughput per unit time and improve production efficiency, and is thought to be industrially useful.
Tanning refers to a process wherein an animal hide/skin is subjected to a series of treatments and steps to render the hide/skin into a durable, flexible leather. This processing is achieved by cross-linking the collagen of the hide/skin with hexavalent chromium. Because hexavalent chromium is harmful and the release thereof into the environment is unwanted, there is a strong demand for the development of an alternative method. Regarding the utilization of transglutaminase for tanning, U.S. Pat. No. 6,849,095 discloses that a transglutaminase of microbial origin can be used for tanning, but discloses no examples of actually allowing the transglutaminase to act on a hide/skin; a transglutaminase has not yet been practically applied for this purpose. Because cross-linking with hexavalent chromium takes place at pH 3 to 4, transglutaminase should also be able to react at this pH, but because MTG is labile to acidity, actual application is difficult.
Hence, when used in applications such as textile processing and tanning, the thermal stability (i.e., heat resistance) of transglutaminase is improved so that the reaction is completed at a high temperature in a short time, and the pH stability is improved so that the reaction can occur under acidic conditions.
As stated above, as a means for modifying and improving the enzymatic activity of transglutaminase, in addition to investigating reaction conditions, modifications of the transglutaminase itself, that is, improvement of the thermal stability and pH stability of the transglutaminase and the like can be mentioned. For example, improving the thermal stability broadens the applicability, which leads to the expectation of increased reaction rates and the like. Also, improving the pH stability will allow the enzymatic reaction to occur under a broader range of pH values, as well as improving the storage stability. This will also be advantageous in industrialization.
To modify the heat resistance and/or pH stability of MTG, it is necessary to prepare a mutant of the MTG, evaluate the activity and the like thereof, and screen for an excellent mutant, that is, a mutant with improved heat resistance and/or pH stability. To prepare a mutant, it is necessary to manipulate the wild-type gene; therefore, a genetically recombinant protein can be prepared. In the case of MTG, a secretory expression system using Corynebacterium glutamicum is known (WO2002/081694).
Secretory expression systems of Corynebacterium are known as the Sec system and the Tat system. In the Sec system, a protein is secreted prior to formation of a higher structure, whereas the Tat system is characterized in that a protein is secreted through the cell membrane after forming a higher structure in the cell (J. Biol. Chem. 25; 273(52): 34868-74, 1998). The Sec system occurs widely, from prokaryotic organisms such as Escherichia coli and Bacillus subtilis, to yeast, fungi, and even to eukaryotic organisms such as humans, and is the most important and most general protein secretion pathway. The Tat system also makes it possible to efficiently secrete a heterogeneous protein, which is difficult to secrete with the Sec system (WO2005/103278). Because MTG can be secreted, whether the Sec system or the Tat system is used, secretion with the Tat system can be attempted if a modification has inhibited secretion with the Sec system.
To increase the stability of a protein, it is generally possible to use a method wherein a non-covalent bond, such as a hydrogen bond, an electrostatic interaction, or a hydrophobic interaction, or a covalent bond, such as a disulfide bond, is introduced to enhance the packing of the hydrophobic core in the molecule, or to stabilize the α helix in the secondary structure. Alternatively, another method which can be used to increase the stability of a protein is to remove a factor that makes the structure of the protein unstable. To increase the stability of a protein by introducing a disulfide bond, it is necessary to find a position suitable for introducing cysteine.